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PH Domain (ph + domain)
Selected AbstractsExpression of PI(4,5)P2 -binding proteins lowers the PI(4,5)P2 level and inhibits Fc,RIIA-mediated cell spreading and phagocytosisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2008Ewelina Szyma Abstract We found that Fc,RII-mediated cell spreading and phagocytosis were correlated with an increase of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] level in cells. During the spreading, a long-lasting elevation of PI(4,5)P2 and concomitant actin polymerization occurred. Filopodia and lamellae of spreading cells were enriched in phosphatidylinositol 4-phosphate 5-kinase I, (PIP5-kinase I,) that colocalized with PI(4,5)P2 and actin filaments. Both spreading and phagocytosis were inhibited by expression of the C374,440 fragment of PIP5-kinase I, or the pleckstrin homology domain of phospholipase C,1 (PLC,1 -PH), two probes binding PI(4,5)P2. These probes reduced the amount of PI(4,5)P2 in the cells, evoked reorganization of the actin cytoskeleton and abolished PI(4,5)P2 elevation during phagocytosis. Simultaneously, PLC,1 -PH-GFP reduced the amount of PIP5-kinase I, associated with the plasma membrane. In vitro studies demonstrated that PIP5-kinase I,-GST bound PI(4,5)P2, phosphatidylinositol 4-monophosphate, and less efficiently, phosphatidic acid. The data suggest that the PLC,1 -PH domain, and possibly also the C374,440 fragment, when expressed in cells, can compete with endogenous PIP5-kinase I, for PI(4,5)P2 binding in the plasma membrane leading eventually to PI(4,5)P2 depletion. [source] PDK1 and PKB/Akt: Ideal Targets for Development of New Strategies to Structure-Based Drug DesignIUBMB LIFE, Issue 3 2003Thomas Harris Abstract Growth factor binding events to receptor tyrosine kinases result in activation of phosphatidylinositol 3-kinase (PI3K), and activated PI3K generates the membrane-bound second messengers phosphatidylinositol 3,4-diphosphate [PI(3,4)P2] and PI(3,4,5)P3, which mediate membrane translocation of the phosphoinositide-dependent kinase-1 (PDK1) and protein kinase B (PKB, also known as Akt). In addition to the kinase domain, PDK1 and PKB contain a pleckstrin homology (PH) domain that binds to the second messenger, resulting in the phosphorylation and activation of PKB by PDK1. Recent evidence indicates that constitutive activation of PKB contributes to cancer progression by promoting proliferation and increased cell survival. The indicating of PDK1 and PKB as primary targets for discovery of anticancer drugs, together with the observations that both PDK1 and PKB contain small-molecule regulatory binding sites that may be in proximity to the kinase active site, make PDK1 and PKB ideal targets for the development of new strategies to structure-based drug design. While X-ray structures have been reported for the kinase domains of PDK1 and PKB, no suitable crystals have been obtained for either PDK1 or PKB with their PH domains intact. In this regard, a novel structure-based strategy is proposed, which utilizes segmental isotopic labeling of the PH domain in combination with site-directed spin labeling of the kinase active site. Then, long-range distance restraints between the 15N-labeled backbone amide groups of the PH domain and the unpaired electron of the active site spin label can be determined from magnetic resonance studies of the enhancement effect that the paramagnetic spin label has on the nuclear relaxation rates of the amide protons. The determination of the structure and position of the PH domain with respect to the known X-ray structure of the kinase active site could be useful in the rational design of potent and selective inhibitors of PDK1 and PKB by 'linking' the free energies of binding of substrate (ATP) analogs with analogs of the inositol polar head group of the phospholipid second messenger. The combined use of X-ray crystallography, segmental isotopic and spin labeling, and magnetic resonance studies can be further extended to the study of other dynamic multidomain proteins and targets for structure-based drug design. IUBMB Life, 55: 117-126, 2003 [source] Serum and glucocorticoid-regulated protein kinases: Variations on a themeJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2006Maude Tessier Abstract The phosphatidylinositol 3, kinase (PI3K)-signaling pathway plays a critical role in a variety of cellular responses such as modulation of cell survival, glucose homeostasis, cell division, and cell growth. PI3K generates important lipid second messengers,phosphatidylinositides that are phosphorylated at the 3, position of their inositol ring head-group. These membrane restricted lipids act by binding with high affinity to specific protein domains such as the pleckstrin homology (PH) domain. Effectors of PI3K include molecules that harbor such domains such as phosphoinositide-dependent kinase (PDK1) and protein kinase B (PKB), also termed Akt. The mammalian genome encodes three different PKB genes (,, ,, and ,; Akt1, 2, and 3, respectively) and each is an attractive target for therapeutic intervention in diseases such as glioblastoma and breast cancer. A second family of three protein kinases, termed serum and glucocorticoid-regulated protein kinases (SGKs), is structurally related to the PKB family including regulation by PI3K but lack a PH domain. However, in addition to PH domains, a second class of 3, phosphorylated inositol phospholipid-binding domains exists that is termed Phox homology (PX) domain: this domain is found in one of the SGKs (SGK3). Here, we summarize knowledge of the three SGK isoforms and compare and contrast them to PKB with respect to their possible importance in cellular regulation and potential as therapeutic targets. J. Cell. Biochem. © 2006 Wiley-Liss, Inc. [source] Rga2 is a Rho2 GAP that regulates morphogenesis and cell integrity in S. pombeMOLECULAR MICROBIOLOGY, Issue 4 2008Ma Antonia Villar-Tajadura Summary Schizosaccharomyces pombe Rho2 GTPase regulates ,-D-glucan synthesis and acts upstream of Pck2 to activate the MAP kinase pathway for cell integrity. However, little is known about its regulation. Here we describe Rga2 as a Rho2 GTPase-activating protein (GAP) that regulates cell morphology. rga2+ gene is not essential for growth but its deletion causes longer and thinner cells whereas rga2+ overexpression causes shorter and broader cells. rga2+ overexpression also causes abnormal accumulation of Calcofluor-stained material and cell lysis, suggesting that it also participates in cell wall integrity. Rga2 localizes to growth tips and septum region. The N-terminal region of the protein is required for its correct localization whereas the PH domain is necessary exclusively for Rga2 localization to the division area. Also, Rga2 localization depends on polarity markers and on actin polymerization. Rga2 interacts with Rho2 and possesses in vitro and in vivo GAP activity for this GTPase. Accordingly, rga2, cells contain more ,-D-glucan and therefore partially suppress the thermosensitivity of mok1,664 cells, which have a defective ,-D-glucan synthase. Additionally, genetic interactions and biochemical analysis suggest that Rga2 regulates Rho2,Pck2 interaction and might participate in the regulation of the MAPK cell integrity pathway. [source] Purification, crystallization and preliminary X-ray diffraction of a proteolytic fragment of PDK1 containing the pleckstrin homology domainACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2004David Komander 3-Phosphoinositide-dependent protein kinase-1 (PDK1) is a Ser/Thr kinase with an essential role in insulin and growth-factor signalling. PDK1 activity towards protein kinase B (PKB) is partially regulated by its pleckstrin homology (PH) domain, which preferentially binds to 3-phosphoinositides. However, the precise molecular mechanism of this regulation is not well understood. Here, the cloning, purification and crystallization of a 150-amino-acid C-terminal region of PDK1 containing the PH domain is reported. A crystal of the PDK1 PH domain grown in the presence of inositol 1,3,4,5-tetrakisphosphate and derivatized with AuCN diffracted to 1.5,Å at a synchrotron source. Diffraction data collected near the Au edge resulted in an anomalous Patterson map with a 30, peak. [source] Rab5a overexpression promoting ovarian cancer cell proliferation may be associated with APPL1-related epidermal growth factor signaling pathwayCANCER SCIENCE, Issue 6 2010Zhen Zhao Rab5a is a regulatory guanosine triphosphatase that is associated with the transport and fusion of endocytic vesicles, and participates in regulation of intracellular signaling pathways embraced by cells to adapt to the specific environment. Rab5a is also correlated with lung, stomach, and hepatocellular carcinomas. Here, we detected Rab5a in paraffin-embedded samples of 20 ovarian cysts, 20 benign cystadenomas, and 39 ovarian cancers by immunohistochemistry, and observed that Rab5a expression was significantly higher in ovarian cancer (P = 0.0001). By setting up stable HO-8910 cell lines expressing Rab5a or dominant negative Rab5a (Rab5a:S34N), we found that Rab5a overexpression enhanced the cell growth by promoting G1 into S phase. In contrast, Rab5a:S34N inhibited this process. Additionally, APPL1 (adaptor protein containing PH domain, PTB domain, and Leucine zipper motif), a downstream effector of Rab5a, was also involved in promoting HO-8910 cell cycle progress. But this function was blocked by Rab5a:S34N. Laser scanning confocal microscopy represented the colocalization of APPL1 and Rab5a in the plasmolemma, which changed with the time of epidermal growth factor (EGF) stimulation. We also found APPL1 could transfer from the membranes into the nucleus where it interacted with NuRD/MeCP1 (the nucleosome remodeling and histone deacetylase multiprotein complex). NuRD is reported to be involved in the deacetylation of histone H3 and H4 to regulate nuclear transcription. So Rab5a promoted proliferation of ovarian cancer cells, which may be associated with the APPL1-related epidermal growth factor signaling pathway. (Cancer Sci 2010) [source] Serum and glucocorticoid-regulated protein kinases: Variations on a themeJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2006Maude Tessier Abstract The phosphatidylinositol 3, kinase (PI3K)-signaling pathway plays a critical role in a variety of cellular responses such as modulation of cell survival, glucose homeostasis, cell division, and cell growth. PI3K generates important lipid second messengers,phosphatidylinositides that are phosphorylated at the 3, position of their inositol ring head-group. These membrane restricted lipids act by binding with high affinity to specific protein domains such as the pleckstrin homology (PH) domain. Effectors of PI3K include molecules that harbor such domains such as phosphoinositide-dependent kinase (PDK1) and protein kinase B (PKB), also termed Akt. The mammalian genome encodes three different PKB genes (,, ,, and ,; Akt1, 2, and 3, respectively) and each is an attractive target for therapeutic intervention in diseases such as glioblastoma and breast cancer. A second family of three protein kinases, termed serum and glucocorticoid-regulated protein kinases (SGKs), is structurally related to the PKB family including regulation by PI3K but lack a PH domain. However, in addition to PH domains, a second class of 3, phosphorylated inositol phospholipid-binding domains exists that is termed Phox homology (PX) domain: this domain is found in one of the SGKs (SGK3). Here, we summarize knowledge of the three SGK isoforms and compare and contrast them to PKB with respect to their possible importance in cellular regulation and potential as therapeutic targets. J. Cell. Biochem. © 2006 Wiley-Liss, Inc. [source] The cell migration protein Grb7 associates with transcriptional regulator FHL2 in a Grb7 phosphorylation-dependent mannerJOURNAL OF MOLECULAR RECOGNITION, Issue 1 2009Sharareh Siamakpour-Reihani Abstract Grb7 is an adaptor molecule that can mediate signal transduction from multiple cell surface receptors to various downstream signaling pathways. Grb7, along with Grb10 and Grb14, make up the Grb7 protein family. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7 and a receptor tyrosine kinase (RTK), erbB2, are overexpressed in 20,30% of breast cancers. Grb7 overexpression has been linked to enhanced cell migration and metastasis, though the participants in these pathways have not been determined. In this study, we report that Grb7 interacts with four and half lim domains isoform 2 (FHL2), a transcription regulator with an important role in oncogenesis, including breast cancer. Additionally, in yeast 2-hybrid (Y2H) assays, we show that the interaction is specific to the Grb7 RA and PH domains. We have also demonstrated that full-length (FL) Grb7 and FHL2 interact in mammalian cells and that Grb7 must be tyrosine phosphorylated for this interaction to occur. Immunofluorescent microscopy demonstrates possible co-localization of Grb7 and FHL2. A model with supporting NMR evidence of Grb7 autoinhibition is proposed. Copyright © 2008 John Wiley & Sons, Ltd. [source] | ||||||||||||||||